The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. The following major findings were made over the last year: 1) We have recently established that the mitochondria forms a reticulum across the skeletal muscle cell that permits the rapid transmission of potential energy for muscle contraction across the cell. This is critical for the fundamental energy support of muscle contraction. We published this year the mitochondrial reticulum structure of the heart cell which was distinctly different from the skeletal muscle cell. In the heart this network coupled more tightly across the long axis of the muscle rather than the short axis as in the skeletal muscle. We reasoned this to be due to the reduced diameter of the heart cell when compared to the heart muscle. We are currently attempting to look for the proteins associated with the formation of the mitochondria reticulum in heart cells. 2) This tight coupling of mitochondria across all of the muscle cells is also a risk. If one mitochondria fails, it could pull down the entire mitochondrial network just like a short circuit in a house. We have recently published (Power Grid Protection of the Muscle Mitochondrial Reticulum see bibliography) that a rapid fail safe system is in place that rapidly removed damaged mitochondria from the network. Our current hypothesis is that this fail safe, or circuit breaker, mechanism is structural in nature representing the physical uncoupling of the mitochondria from the network. This rapid distribution of energy within the muscle cell contrasts with earlier models relying on slow high energy metabolite diffusion and provides another parameter to evaluate in different clinical conditions. The mechanisms associated with this rapid removal of mitochondria from the network is an ongoing effort in the laboratory. 3) We have also published a modeled (The electrochemical transmission in I-Band segments of the mitochondrial reticulum: see bibliography) on how the electrical conduction occurs across the mitochondrial reticulum and reached the conclusion that the dominate ions, such as potassium, sodium and chloride must carry this current with appropriate proton-ion exchange systems. 4) Using our ability to monitor subcellular events rapidly in the living animal, we have initiated a collaboration with Dr. Sinnis at Johns Hopkins to monitor the trafficking of malaria parasites upon inject via a simulated mosquito proboscis. The simulated proboscis is a specially designed fluorescent glass pipet that we can monitor in the animal with a 2-Photon excitation microscope. Using genetically labeled parasites with green fluorescent protein and mice ( with a vessel wall fluorescent protein) we have been able to observe the first few seconds of the inoculation process and observe how the parasites behave under the skin. We hope these studies will reveal how the parasites find and penetrate blood vessels to initiate the malaria infection. This understanding may provide a new strategy in preventing the malaria infection immediately after the inoculation.